Chondrocyte Therapeutic Delivery Stystem

ABSTRACT

Methods and compositions for producing therapeutic agents using chondrocytes are provided. The genetically engineered chondrocytes can be used to express the therapeutic agent in a subject, including in an environment typically associated with chondrocytes and in an environment not typically associated with chondrocytes.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.10/657,516 filed on Sep. 8, 2003 and entitled “Chondrocyte TherapeuticDelivery System,” which is hereby incorporated by reference in itsentirety.

FIELD OF THE INVENTION

This invention relates to the treatment or amelioration of disorders ordiseases using chondrocytes to deliver therapeutic agents.

BACKGROUND OF THE INVENTION

Chondrocytes are typically involved in cartilage repair. Cartilage is astructural support tissue that is found in the body in three mainvarieties. Hyaline, or articular cartilage, helps dissipate loads injoints. In articular cartilage, chondrocytes are encapsulated in awoven, mesh-like matrix of type II collagen and proteoglycans. Elasticcartilage provides flexible support to external structures, and iscomposed of chondrocytes embedded in a matrix of collagen and elasticfibers. Fibrocartilage aids in transferring loads between tendons andbone. It consists of an outer layer of collagen and fibroblasts thatsupport and inner layer of chondrocytes that make type II collagenfibers.

To date chondrocytes have been used to correct or repair cartilaginousdefects. For example, by placing chondrocytes into hydrogels andinjecting the chondrocytes into a cartilage defective region. Thechondrocytes are used to express matrix proteins required for cartilagerepair. Although chondrocytes have been cultured and placed intosubstrates such as hydrogels, for treatment, or repair of cartilage orbone defects, there is no teaching in the art that chondrocytes can beused to express therapeutic agents for the treatment of pathologies orinjuries other than cartilage tissue.

Accordingly, a need exists for using chondrocytes to deliver therapeuticagents to tissue or organs within the body, and particularly to anenvironment that is not usually associated with chondrocytes. A needalso exists for using an implant comprising genetically alteredchondrocytes that can locally express and release therapeutic agentsinto a target region or environment. A further need exists for usingchondrocytes as a large scale manufacturing source for therapeuticagents.

SUMMARY OF THE INVENTION

The present invention provides methods and compositions for usinggenetically altered chondrocytes as vehicles to express therapeuticagents. The methods of the invention are particularly conducive to theuse of chondrocytes for expressing a therapeutic agent in localizedenvironments that are not typically associated with chondrocytes.Specifically, the genetically altered chondrocytes can be used toexpress therapeutic agents, such as proteins or antibodies, intolocalized environments such as the central nervous system (e.g., thebrain or spinal cord) or into solid organs (e.g., heart, liver orkidney). This type of localized delivery and expression of thetherapeutic agent provides a localized concentration of the therapeuticagent in a particular region, without the necessary side effects thatresult from systemic administration of the therapeutic agent. Thegenetically altered chondrocytes can be introduced into theseenvironments, using suitable matrix substrates, such as biological gels(e.g., hydrogels).

Chondrocytes offer several unique advantages as vehicles for expressingtherapeutic agents over other cell types. For example, chondrocytes donot require vascular support, therefore can readily be used inenvironments that have a reduced, or non-existent vascularizationsystem. Furthermore, chondrocytes are able to survive in harsh in vivoenvironments, including ischemic tissue, as well as low pH and lowoxygen surroundings. In addition, there is a reduced likelihood ofmalignancy due to the anti-angiogenicity properties of normalchondrocytes. Chondrocytes also possess an immune privileged propertywhich reduces immune rejection of co-implanted allogenic or xenogenictissue. Furthermore, chondrocytes are more easily scalable compared toother normal untransformed cell strains.

The genetically altered chondrocyte can be used to deliver and expresstherapeutic agents in an environment that requires modification with thetherapeutic agent. In one embodiment, the genetically alteredchondrocytes can be used to express a therapeutic agent in anenvironment associated with chondrocytes, such as the cartilage,tendons, joints, and bones. When the genetically engineered chondrocytesare used to express therapeutic agents in environments associated withchondrocytes, they are used for the purpose expressing the therapeuticagent, they do not become part of the structural component of theenvironment, i.e., they are not used as a component of a tissueengineered construct to repair or replace a damaged cartilage, tendon,joint, or bone. In another embodiment, the genetically alteredchondrocytes are used to produce a therapeutic agent at an ectopic site,that is, in an environment that is not typically associated withchondrocytes such as the brain, or solid organs (e.g., heart, kidney,liver). Other atypical chondrocyte environments include an aqueousenvironment such as blood, and plasma.

The genetically altered chondrocyte can be mixed with a biocompatiblesubstrate, such as a gel matrix substrate or another scaffold matrixmade of natural or synthetic material. This genetically alteredchondrocyte-gel matrix substrate or chondrocyte-scaffold matrix can beinjected to a target region. Alternatively, the genetically alteredchondrocyte-gel matrix substrate or chondrocyte-scaffold matrix can beprepared prior to implantation to a target region and surgicallyimplanted into an ectopic site. The genetically altered chondrocytes canbe used to treat or ameliorate a number of disorders by modifying thecell associated with the disorder by expressing the therapeutic agent.The cells associated with a disorder include disorders such as a blooddisorder; a cardiovascular disorder; an endocrine disorder; anautoimmune disorder; a neurological disorder; a skin disorder; bonedisorder (osteoporosis); a fertility disorder; a metabolic disorder oruncontrolled growth disorder such as cancer. In other embodiments, thegenetically altered chondrocytes can also be used to address conditionsthat are not associated with a disorder, for example, to express atherapeutic agent such as hormones for birth control and reproduction.

Accordingly, in one aspect, the invention pertains to a geneticallyaltered chondrocyte used for expressing a therapeutic agent. Thegenetically altered chondrocyte is delivered to a cell associated with adisorder and expresses the therapeutic agent to modify an environmentsurrounding the cell. However, the genetically altered chondrocyte isnot structurally functional the environment surrounding the cell.

In one embodiment, the cell associated with a disorder can be a cell inan atypical chondrocyte environment such as an organ selected from thegroup consisting of the brain, heart, liver, kidney, gastro-intestinaltract, spleen, smooth muscles, skeletal muscles, eye, ganglions, lungs,gonads, and pancreas. With organs, the genetically altered chondrocytecan be delivered to one or more regions of the organ. The atypicalchondrocyte environment can also be an aqueous environment selected fromthe group consisting of blood, plasma, the vitreous humor of the eye,spinal fluid, and the like. The genetically altered chondrocyte can bedelivered to the spleen, the bone marrow, a ganglion, the peritonealcavity or any other well vascularized tissue for release into the bloodor plasma. In another embodiment, the cell associated with a disorder isin a typical chondrocyte environment such as a bone, tendon andcartilage.

The genetically altered chondrocyte can be delivered on their own or ina mixture with a biocompatible substrate. The biocompatible,bioabsorbable substrate can be a substantially solid polymeric materialor it can be in the form of a gel matrix. In a preferred embodiment, thebiocompatible substrate is gel matrix substrate.

The cell associated with a disorder can be a cell selected from thegroup consisting of a cell associated with a blood disorder, a cellassociated with a cardiovascular disorder, a cell associated with anendocrine disorder, a cell associated with an autoimmune disorder, acell associated with a neurological disorder, a cell associated with askin disorder, a cell associated with fertility disorder, and cellassociated with reproduction.

The genetically altered chondrocyte can also be used to express atherapeutic agent in a target region associated with a disorder bydelivering (e.g., ectopically) the genetically altered chondrocyte to atleast one target region. The therapeutic agent that is expressed in thetarget region can either modify the target region alone (e.g., localizedtargeting of a target region, e.g., a knee joint). Alternatively, thegenetically altered chondrocytes can be used to express a therapeuticagent in the target region, but the therapeutic agent can modify theenvironment surrounding the target region (e.g., using chondrocytes toexpress insulin in the pancreas, where the expressed insulin effects thesystemic blood glucose concentration).

Accordingly, in another aspect, the invention pertains to a geneticallyaltered chondrocyte used for expressing a therapeutic agent in a targetregion associated with a disorder, wherein the genetically alteredchondrocyte is delivered to the target region and expresses thetherapeutic agent to modify the target region or an environmentsurrounding the target region, and wherein the genetically alteredchondrocyte is not structurally functional in the target region or theenvironment surrounding the target region.

In yet another aspect, the invention pertains to a method for modifyingan environment of a cell associated with a disorder using a geneticallyaltered chondrocyte, by delivering a genetically altered chondrocyte tothe cell associated with the disorder, wherein the genetically alteredchondrocyte has been altered to express a therapeutic agent, and whereinthe genetically altered chondrocyte is not structurally functional inthe environment surrounding the cell. The therapeutic agent is thenexpressed at a level sufficient to modify the environment surroundingthe cell. The environment can be one associated with a chondrocyte underthe provision that the genetically altered chondrocyte is used only toexpress the therapeutic agent. The genetically altered chondrocyte isnot structurally functional within the structure of the environment. Ina preferred embodiment, the environment is chondrocyte atypicalenvironment, such as the brain, heart, liver, kidney, pancreas, skeletalmuscle, skin, spinal cord, spleen, eye, peritoneal cavity,gastro-intestinal track, or regions thereof.

In yet another embodiment, the invention pertains to a method forameliorating a disorder in a subject using a genetically alteredchondrocyte, by implanting a biocompatible substrate comprising agenetically altered chondrocyte into a target region of the subject,wherein the genetically altered chondrocyte has been altered to producea therapeutic agent, and wherein the genetically altered chondrocyte isnot structurally functional in the target region or an environmentsurrounding the target region. The therapeutic agent is then expressedin the target region at a level sufficient to ameliorate the disorder.

The biocompatible substrate that is implanted can be a gel matrixsubstrate, such as an alginate, polysaccharide, and agarose. Thisgenetically altered chondrocyte-gel matrix substrate can be injected toa target region. Alternatively, the genetically altered chondrocyte-gelmatrix substrate can be solidified prior to implantation to a targetregion. When the chondrocyte-gel matrix substrate can be solidifiedprior to implantation, the dimensions of the implanted gel matrixsubstrate determine the concentration of chondrocytes within the gelmatrix substrate that are available to express the therapeutic agent.The concentration of chondrocytes in the gel matrix substrate is in therange of about 10,000 to hundreds of millions of cells per ml of gelmatrix having a volume in the range of about 0.1 to 10 ml. Preferably,the concentration of the chondrocytes is about 100,000 to about onemillion cells per ml of substrate. Preferably, the volume of the gelmatrix substrate is less than one milliliter. It is understood that thedose necessary to treat a particular pathology could be easily optimizedby either changing the cellular concentration for a given allowablevolume, or by changing the amount of chondrocyte-gel implanted. Theinjury to be ameliorated can be a wound, a bone defect, a cartilagedefect, a skin wound, and a torn ligament. The disorder that can beameliorated can be a blood disorder, an autoimmune disorder, a hormonaldisorder, an anti-inflammatory disorder, a fertility disorder, and anneurodegenerative disorder.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a photograph of cultured bovine chondrocytes;

FIG. 2A is a phase contrast photograph of chondrocytes transfected withGreen Fluorescent Protein (GFP);

FIG. 2B is a chondrocyte from FIG. 2A showing expression of GreenFluorescent Protein (GFP);

FIG. 3A is a graph showing expression of EPO from human chondrocytes;

FIG. 3B is a graph showing expression of EPO from bovine chondrocytes;

FIG. 4 is a graph showing expression of EPO mimetibody from humanchondrocytes;

DETAILED DESCRIPTION OF THE INVENTION

The practice of the present invention employs, unless otherwiseindicated, conventional methods of virology, microbiology, molecularbiology and recombinant DNA techniques within the skill of the art. Suchtechniques are explained fully in the literature. (See, e.g., Sambrook,et al. Molecular Cloning: A Laboratory Manual (Current Edition); DNACloning: A Practical Approach, Vol. I & II (D. Glover, ed.);Oligonucleotide Synthesis (N. Gait, ed., Current Edition); Nucleic AcidHybridization (B. Hames & S. Higgins, eds., Current Edition);Transcription and Translation (B. Hames & S. Higgins, eds., CurrentEdition); CRC Handbook of Parvoviruses, Vol. I & II (P. Tijessen, ed.);Fundamental Virology, 2nd Edition, Vol. I & II (B. N. Fields and D. M.Knipe, eds.)).

So that the invention may more readily be understood, certain terms arefirst defined:

The term “chondrocyte” as used herein refers to the art recognized useof the term for a cell type involved in cartilage formation and repair.The chondrocyte functions to produce extracellular matrix ofproteoglycans and collagen. Also included with in the use of the term“chondrocyte” are chondrocyte precursor cells such as chondroblasts, andmesenchymal stem cells.

The term “genetically altered chondrocyte” as used herein refers to achondrocyte cell that has been manipulated using standard molecularbiological techniques, in a way that introduces a heterologous orforeign nucleic acid molecule encoding a therapeutic agent into thecell.

The term “nucleic acid” sequence as used herein refers to a DNA or RNAsequence. The term captures sequences that include any of the known baseanalogues of DNA and RNA such as, but not limited to, 4-acetylcytosine,8-hydroxy-N6-methyladenosine, aziridinylcytosine, pseudoisocytosine,5-(carboxyhydroxylmethyl) uracil, 5-fluorouracil, 5-bromouracil,5-carboxymethylaminomethyl-2-thiouracil,5-carboxymethylaminomethyluracil, dihydrouracil, inosine,N6-isopentenyladenine, 1-methyladenine, 1-methylpseudo-uracil,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-methyladenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarbonylmethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid methylester,uracil-5-oxyacetic acid, oxybutoxosine, pseudouracil, queosine,2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil,5-methyluracil, N-uracil-5-oxyacetic acid methylester,uracil-5-oxyacetic acid, pseudouracil, 2-thiocytosine, and2,6-diaminopurine.

The term “therapeutic agent” as used herein refers to a compound thatproduces a desired therapeutic effect. The therapeutic agent can beselected from the group consisting of a protein, an agonist or anantagonist of an antibody, an antigen, a hormone, an anti-inflammatoryagent, an antiviral agent, an anti-bacterial agent, a growth factor, acytokine, a hormone, an oncogene, a tumor suppressor, a transmembranereceptor, a protein receptor, a serum protein, an adhesion molecule, aneurotransmitter, a morphogenetic protein, a differentiation factor, anenzyme, matrix proteins, and an extracellular matrix protein, iRNA, RNA,or fragment and peptides thereof. In a preferred embodiment, thetherapeutic agent is protein such as the Erythropoietin (EPO) protein.Other examples of suitable proteins include, but are not limited to,insulin protein, pro-insulin protein, Remicade, bone morphogeneticprotein (BMPs), Transforming growth factor-beta (TGF-beta),Platelet-derived growth factor (PDGF), cartilage derived morphogenicprotein (CDMP), and MP-52.

In another embodiment, the therapeutic agent is an antibody, an antibodyfragment, or a mimetibody. Examples of a useful mimetibody include butare not limited to EPO mimetibody, Remicade mimetibody, BMP mimetibody,cartilage derived morphogenic protein (CDMP) mimetibody and MP-52. In apreferred embodiment, the antibody is the EPO mimetibody.

In yet another embodiment, the therapeutic agent is a growth factor.Preferred growth factors include, but are not limited to, epidermalgrowth factor, bone morphogenetic protein, vascular endothelial-derivedgrowth factor, hepatocyte growth factor, platelet-derived growth factor,hematopoetic growth factors, heparin binding growth factor, peptidegrowth factors, and basic and acidic fibroblast growth factors. In someembodiments it may be desirable to incorporate genes for factors such asnerve growth factor (NGF) or muscle morphogenic factor (MMP), TGF-betasuperfamily, which includes BMPs, CDMP, and MP-52. In yet anotherembodiment, the therapeutic agent is a receptor. Examples of receptorsinclude, but are not limited to, EPO Receptor, B Cell Receptor, FasReceptor, IL-2 Receptor, T Cell Receptor. EGF Receptor, Wnt, InsulinReceptor, TNF Receptor.

The terms “modifies” or “modified” “modifying” or “modification” areused interchangeably herein and refer to the up-regulation ordown-regulation of the therapeutic agent in a cell associated with adisorder, or at a target region. For example, the genetically modifiedchondrocyte can be used to express a therapeutic agent associated with ablood disorder, e.g., the EPO protein, by delivering the geneticallyaltered chondrocyte to the liver or kidney, or any tissue or organ witha vascular supply to allow EPO to reach the target site or region. Oncethe EPO protein is expressed, it can enter the circulatory system andbind to, and alter the function of the EPO receptor (EPOR). This in turnwill cause a change in the environment associated with the EPO receptor,for example by modifying the signal transduction cascade involving theEPO receptor. Thus, the term “modifies” or “modified” also refers to theincrease, decrease, elevation, or depression of processes or signaltransduction cascades involving the therapeutic agent (e.g., EPO), or aprotein associated with the therapeutic agent (e.g., EPOR).

Modification of the cell may occur directly, for example byoverexpressing EPO in a target region. Alternatively, modification of acell may occur indirectly, for example by the overexpressed EPOinteracting with an EPOR that lead to a changes in downstream signaltransduction cascades involving the EPOR. Non-limiting examples ofmodifications include cell proliferation response, cell differentiation,modifications of morphological and functional processes, under- orover-production or expression of a substance or substances by a cell,e.g., a hormone, growth factors, etc, failure of a cell to produce asubstance or substances which it normally produces, production ofsubstances, e.g., neurotransmitters, and/or transmission of electricalimpulses.

These terms are also used to describe the effect of the expressedtherapeutic agent on the cell, the target region, or the environmentsurrounding the cell or target region. The therapeutic agent isexpressed at a level sufficient to produce a desired therapeutic effectby altering the cell, target region, or the environment surrounding thecell or target region. For example, the expression of EPO in a targetregion, (e.g., the liver), will produce elevated levels of EPO thatalter blood hematocrit, and hemoglobin levels.

The phrases “an atypical chondrocyte environment,” a “non-chondrocytetypical environment,” “an environment not usually associated withchondrocytes,” and an ectopic site are used interchangeably herein andrefer to a surrounding in which chondrocytes are not present. Examplesof an environment not usually associated with chondrocytes include thecentral nervous system (CNS). The CNS which includes the brain andspinal cord, is generally considered immunoprivileged. Other examples ofenvironments that are not usually associated with chondrocytes includesolid organs. Examples of solid organs include, but are not limited to,the heart, kidney, liver and pancreas. Yet another example of anenvironment not usually associated with chondrocytes, is thereproductive organs. In males, the reproductive organs not associatedwith chondrocytes are for example, the testis, vas deferens, and thelike. In females, the reproductive organs not associated withchondrocytes are, for example, the uterus, fallopian tubes, ovaries andthe like. Other examples of an environment not associated withchondrocytes include skin, a subcutaneous pouch, intramuscular andintraperitoneal space.

The term “biocompatible substrate” or “biocompatible matrix” are usedinterchangeably herein, and refer to a material into, or onto which acell population (e.g., a chondrocyte population) can be deposited. Thematerial is suitable for implantation in a subject, and does not causetoxic or injurious effects once implanted in the subject.

The term “subject” as used herein refers to any living organism capableof eliciting an immune response. The term subject includes, but is notlimited to, humans, nonhuman primates such as chimpanzees and other apesand monkey species; farm animals such as cattle, sheep, pigs, goats andhorses; domestic mammals such as dogs and cats; laboratory animalsincluding rodents such as mice, rats and guinea pigs, and the like. Theterm does not denote a particular age or sex. Thus, adult and newbornsubjects, as well as fetuses, whether male or female, are intended to becovered.

The term “vector” as used herein refers to any genetic element, such asa plasmid, phage, transposon, cosmid, chromosome, artificial chromosome,virus, virion, etc., which is capable of replication when associatedwith the proper control elements and which can transfer gene sequencesbetween cells. Thus, the term includes cloning and expression vehicles,as well as viral vectors.

The term “transfection” as used herein refers to the uptake of foreignDNA by a cell. A cell has been “transfected” when exogenous DNA has beenintroduced inside the cell membrane. Such techniques can be used tointroduce one or more exogenous DNA moieties, such as a nucleotideintegration vector and other nucleic acid molecules, into suitable hostcells. The term captures chemical, electrical, and viral-mediatedtransfection procedures.

Various aspects of the invention are described in more detail in thefollowing subsections:

I. Culturing and Genetically Altering A Chondrocyte Cell Population

One aspect of the invention pertains to culturing and geneticallyaltering chondrocytes such that the genetically altered chondrocytesexpress a therapeutic agent. In one embodiment, the genetically alteredchondrocytes are used to produce a therapeutic agent in an environmentthat is not typically associated with chondrocytes, such as the brain,or solid organs (e.g., heart, kidney, liver). In another embodiment, thegenetically altered chondrocytes are used to express a therapeutic agentin an environment associated with chondrocytes, such as the cartilage,tendons, joints, and bones. In such instances, the genetically alteredchondrocytes are used exclusively for manufacturing and expressing thetherapeutic agent, and not as components of a tissue engineeredconstruct used to repair or replace a damaged cartilage, tendon, joint,or bone.

There are a number of reasons for using genetically alteredchondrocytes. To begin with, chondrocytes have the ability to survive invery harsh conditions such as environments with a low pH, or low oxygentension. Thus, the genetically altered chondrocyte population has apost-implantation survival advantage, especially when introduced intodiseased or injured tissues such as stroke-damaged tissue.

Furthermore, the genetically altered chondrocytes require no vascularsupport for survival. This is an important since chondrocytes have abetter chance at surviving encapsulation procedures, such as within agel matrix, as well as implantation procedures. In the past, the sizeand geometry of cell containing capsules have been limited because mostcells depend upon close proximity to vascularity for survival. That is,cells encapsulated in too large a format or in large capsules would havecompromised viability because the cells in the center of the implantwould not have adequate gas and nutrient exchange.

In addition, chondrocytes have a uniform phenotype. The tissue fromwhich the cells are isolated is mostly composed of chondrocytes, and itis free of vascular endothelial cells, stromal cells or any other cellthat forms the cartilage tissue. The physical appearance of thecartilage tissue is also very distinct from the surrounding tissue,making dissection very easy. These characteristics allow for easyisolation of pure population normal chondrocytes. Once the isolatedchondrocytes are placed in tissue culture, they behave similarly tofibroblastic cell lines. The fibroblast-like growth is reversible andthe cells can be easily induced to redifferentiate into chondrocyteswhen placed in anchorage independent conditions such as in agarose oralginate gel suspension. Therefore, immortalized transformed cell lines,which are known to be highly metabolic, with a resultant tendencytowards cancerous mutation, are not necessary.

Chondrocytes typically secrete an extensive extracellular matrix whenplaced in anchorage independent conditions This matrix is thought toprotect the cells from direct cell-cell contact from the blood supply,thereby delaying or preventing immune rejection. This could provide yetanother survival advantage and enable an allogenic or xenogenic implantapproach. Allogenic and xenogenic cells could also have a more simpleregulatory pathway since they could be seen as temporary, and thereforecloser in concept to a device than a permanent tissue transplant.

Furthermore, when the chondrocytes are placed in suspension duringanchorage independent culture, they do not need to form an organizedtissue to perform because the secretion of extracellular matrix, whichprotects against rejection, occurs at the cellular level. Large scalepreparation of chondrocyte-containing gels could be performed, and thedose of the therapeutic could be adjusted by varying the amount or sizeof gel to be implanted without affecting the overall behavior of theimplant. In other words, a small and large implant would differ only inthe number of encapsulated cells, without affecting the biologicalfunctioning of the cells.

The chondrocytes can be isolated from a variety of sources. For example,embryonic chondrocytes can be isolated from sterna (Leboy et al., (1989)J. Biol. Chem., 264:17281-17286; Sullivan et al., (1994) J. Biol. Chem.,269:22500-22506; and Bohme et al., (1995) Exp. Cell Res., 216:191-198),and vertebra (Lian et al., (1993) J. Cellular Biochem., 52:206-219),limb bud mesenchymal cells in micromass cultures (Roark et al., (1994)Develop. Dynam., 200:103-116; and Downie et al., (1994) Dev. Biol.,162:195), growth plate chondrocytes in monolayer (Rosselot et al.,(1994) J. Bone Miner. Res., 9:431-439; Gelb et al., (1990)Endocrinology, 127:1941-1947; and Crabb et al. (1990) J. Bone MineralRes., 5:1105-1112), or pellet cultures (Kato et al., (1988) Proc. Nat.Acad. Sci., 85:9552-9556) have been used to characterize chondrocyteresponses to exogenous factors, many of which function in an autocrinemanner.

Chondrocytes can be obtained by a biopsy of cartilaginous tissue from anarea treated with local anaesthetic with a small amount of lidocaineinjected subcutaneously. The chondrocyte cells from the biopsied tissuecan be expanded in culture. The biopsy can be obtained using a biopsyneedle, a rapid action needle which makes the procedure quick andsimple. In a preferred embodiment, chondrocytes are isolated from donortissue and large cell banks are carefully characterized and optimized toprovide a uniform product.

Chondrocyte cells obtained by biopsy can be cultured and passaged asnecessary. For example, articular cartilage can be aseptically obtainedfrom calf knee joints. Cartilage can be shaved from the end of the bonesusing a scalpel. The tissue can be finely minced in saline solution andsubmitted to collagenase and trypsin digestion for several hours at 37°C. until the tissue has completely digested and single cells aregenerated. The chondrocyte cells isolated from the cartilage tissue canthen be washed from enzyme with DMEM containing 10% fetal bovine serum,and seeded onto tissue culture plastic dishes using about 5,000 cells toabout 10,000 cells per square centimeter. The chondrocyte cells can becultured in DMEM and 10% fetal calf serum with L-glutamine (292 μg/cc),penicillin (100 U/cc), streptomycin (100 μg/cc) at 37° C. in 5% CO₂ withan atmosphere having 100% humidity and expanded in number (See FIG. 1).The chondrocyte cells can then be removed from the tissue culture vesselusing trypsin/EDTA and passaged into larger tissue culture vessels orencapsulated in gel suspension. Expanded chondrocyte cells can also befrozen according to standard procedures until needed.

Other sources from which chondrocytes can be derived includeadipose-derived cells, mesenchymal stem cells, fetal stem cells,marrow-derived stem cells, and other pluripotent stem cells. Thechondrocytes can be autogeneic, isogeneic (e.g., from an identicaltwin), allogeneic, or xenogeneic.

A number of studies have been conducted on the isolated chondrocytes,from which has emerged the critical role for a number of growth factors,including basic fibroblast growth factor (bFGF), transforming growthfactor beta (TGF□)□, insulin-like growth factor-1 (IGF-1), andparathyroid hormone (PTH), which regulate chondrocyte proliferation anddifferentiation. The expression of these factors and their associatedreceptors are maturation dependent and exquisitely regulated (Bohme, etal., (1992) Prog. Growth Factor Res. 4:45-68). Other studies have shownthat vitamins A, C, and D are also required for chondrocyte maturation(Leboy et al., (1994) Microscopy Res. and Technique 28:483-491; Iwamotoet al., (1993) Exp. Cell Res., 207:413-420; Iwamoto et al., (1993) Exp.Cell Res. 205:213-224; Pacifici et al., (1991) Exp. Cell Res. 195:38-46;Shapiro et al., (1994) J. Bone Min. Res. 9:1229-1237; Corvol et al.(1980) FEBS Lett. 116:273-276; Gerstenfeld et al., (1990) Conn. Tiss.Res. 24:29-39; Schwartz et al., (1989) J. Bone Miner. Res. 4:199-207;and Suda, (1985) Calcif Tissue Int., 37:82-90).

The chondrocyte cells may be engineered to incorporate a nucleic acidencoding a therapeutic agent such as proteins, antibodies, regulatorsand cytokines. Other molecules, genes, or nucleic acids that influencecell growth, matrix production, or other cellular functions such as cellcycle may also be used. Nucleic acids may be DNA, RNA, or othernucleotide polymers. Such polymers may include natural nucleosides,nucleoside analogs, chemically modified bases, biologically modifiedbases, intercalated bases, modified sugars, or modified phosphategroups.

In one embodiment, the nucleic acid can be introduced into thechondrocytes as naked DNA, nanocapsules, microspheres, beads, andlipid-based systems such as liposomes, or carriers such as keyholelimpet hemocyanin (KLH) and human serum albumin (See e.g., U.S. Pat. No.6,303,379, to Selden, incorporated herein by reference).

In another embodiment, the chondrocytes may be transfected using vectorsengineered to carry a nucleic acid that encodes the therapeutic agent.The nucleic acid sequences can be cloned into the vector using standardcloning procedures known in the art, as described by Maniatis et al.,Molecular Cloning: A Laboratory Manual, Cold Springs Laboratory, ColdSprings Harbor, N.Y. (1982), which is hereby incorporated by reference.Suitable vectors include, but are not limited to plasmid vectors such aspRO-EX (Gibco/BRL), pBR322, pBR325, pACYC177, pACYC184, pUC8, pUC9,pUC18, pUC19, pLG339, pR290, pKC37, pKC101, SV 40, pBluescript II SK⁺ orKS⁺ (See Stratagene Cloning Systems Catalog from Stratagene, La Jolla,Calif., which is hereby incorporated by reference), pQE, pIH821, pGEX,pET series (See Studier et al., Use of T7 RNA Polymerase to DirectExpression of Cloned Genes, Gene Expression Technology vol. 185 (1990),which is hereby incorporated by reference) and any derivatives thereof.In a preferred embodiment, the plasmid vector is pCDNA3.1. In anotherpreferred embodiment, the plasmid vector is pCEP4.

The gene encoding the therapeutic agent (e.g., EPO) can be operablylinked to other elements regulating expression of the gene, including,but not limited to, a promoter, an enhancer sequence, repressorsequence, TATA box, transcription stop sequence, ribosomal binding site,post-transcriptional regulatory element, etc. One of skill in the artwould appreciate the variety of elements that may be used in combinationwith the therapeutic gene.

Promoters vary in their “strength” (i.e., their ability to promotetranscription), and can be constitutive. For the purposes of expressinga therapeutic gene, it is desirable to use strong promoters in order toobtain a high level of transcription and expression of the therapeuticgene. In one embodiment, the promoter is a constitutive promoter thatincludes, but is not limited to, CMV, RSV promoters, and the like. Otherpromoters include, but are not limited to, LTR or SV40 promoter, the E.coli. lac or trp, T3 and T7 promoters, HSV thymidine kinase, as well asother promoters known to control expression of genes in prokaryotic oreukaryotic cells. In another embodiment, the promoter is specific for aparticular cell type. For example, a promoter is functional in thecentral nervous system (e.g., glial specific promoters, neuron elonasepromoter, and the like); functional in the organs such as the liver(e.g., albumin and alpha1 antitrypsin promoters, and the like, that areactive in hepatocytes); functional in the pancreas (e.g., insulinpromoter, pancreatic amylase promoter and the like); or functional inthe heart (e.g., ventricular myocyte-specific promoter, alpha-MyHC andbeta-MyHC promoter). One skilled in the art will appreciate that thevectors can be readily engineered to include any desired regulatoryelements that are required for the specific transcription of thetherapeutic gene using standard molecular biology techniques.

Also within the scope of the invention are biologically active fragmentsof the therapeutic agent that produce a desired effect. For example, thecomplete gene for EPO can be transfected into a chondrocyte.Alternatively, active fragments of the gene can be used. Thus, thetransfected gene may code for the entire therapeutic agent, or for abiologically active peptide. Also within the scope of the invention arehomologous proteins that are at least 50, 75, or 90% homologous to thetherapeutic agent. Alternatively, a DNA sequence representing afunctional peptide agonist could also be used.

A variety of gene transfection techniques are known in the art. Thenucleic acid sequence can be introduced into cells via transformation,particularly transduction, conjugation, mobilization, orelectroporation. For example, viral vectors such as adenovirus arecommonly used to insert DNA into a variety of cells. Other transfectionmethods include electroporation, calcium-phosphate methods, and lipidbased methods. (See e.g., Sambrook et al., Molecular Cloning: ALaboratory Manual, 2nd Ed., 1989; Miller and Calos, eds., Gene TransferVectors for Mammalian Cells, 1987; Ausubel et al., eds., CurrentProtocols in Molecular Biology, 1987; each of which is incorporatedherein by reference). Both stable transfection and transient expressiontechniques may be employed, depending on how long the therapeutic geneshould be expressed.

Those chondrocytes that have been genetically altered to carry theplasmid encoding the therapeutic agent, can be selected by any methodavailable to the skilled artisan. In a preferred embodiment, thegenetically altered chondrocytes are selected using an antibioticmarker, such as neomycin. This allows for the selection of only thosechondrocytes that have been genetically altered. These isolatedchondrocytes can then be amplified to provide a population of cells thatcarries the plasmid encoding the therapeutic agent. These alteredchondrocytes retain the phenotype of a typical chondrocytes, butprimarily express the therapeutic agent. These genetically alteredchondrocyte population does not become a structural part of the cell,target region or the environment surrounding the cell or target region.The genetically altered chondrocytes function only to express thetherapeutic agent.

II. Environments and Therapeutic Agents

The invention pertains to using genetically engineered chondrocytes asvehicles to deliver a therapeutic agent to an environment. In oneembodiment, the environment can be one that is not typically associatedwith chondrocytes, such as the central nervous system, or solid organssuch as the heart, liver or kidney. In another embodiment, theenvironment is one associated with chondrocytes, however, thechondrocytes still have the chondrocyte phenotype and they are usedexpressly to produce the therapeutic agent. The chondrocytes do notperform the function of cartilage tissue (enabling friction-freearticulation), and thus they are not used for tissue repair orconstruction with a tissue engineered construct. In one embodiment, thegenetically altered chondrocytes are engineered to express onetherapeutic agent, (e.g., EPO). In another embodiment, the geneticallyaltered chondrocytes are engineered more than one therapeutic agents,for example, two therapeutic agents, three therapeutic agents, or morethan three therapeutic agents.

The genetically altered chondrocytes can be delivered to a cell ortarget region using known procedures for delivering vectors. Forexample, the genetically altered chondrocytes can be mixed with a liquidgel and injected to a target region using a surgical syringe. The liquidgel can solidify in situ at the target region. Alternatively, achondrocyte-gel matrix solid can be surgically implanted into a targetregion. The gel matrix solid can be in the form of a thin sheet that canbe rolled or folded, and inserted into the target region using asurgical instrument, and allowed to unfold at the target region (e.g.,interocular lens). The genetically altered chondrocytes alone (i.e.,without being mixed with a substrate), can be injected to a targetregion.

Examples of environments not typically associated with chondrocytesinclude, but are not limited to the following:

(i) Central Nervous System

The genetically altered chondrocytes of the invention can be used toameliorate or modify the function of cells associated with aneurodegenerative or neurological disorder involving the central nervoussystem. Regions of the central nervous system that can be targeted bythe genetically altered chondrocytes of the invention include the cellsin the brain and the spinal cord. Examples of therapeutic agents thatcan be expressed by genetically altered chondrocytes in the centralnervous system include, but are not limited to, receptors (e.g.N-methyl-D-aspartate (NMDA) receptor, GluR receptor (e.g., GluR4,GluR6)); neurotransmitters (e.g., dopamine, acetylecholine, andnorepharine); transporters (e.g., glutamate transporters such asexcitatory amino acid transporters (EAAT)); growth factors (e.g.,neurotrophic factor (GDNF), ciliary derived neuronotrophic factor(CNTF), brain derived neuronotrophic factor (BDNF), neuronotrophin-3(NT3), epidermal growth factor (EGF), fibroblast growth factor (FGF),transforming growth factor-a (TGF-a), transforming growth factor-b(TGF-b), platelet derived growth factor (PDGF)).

a) Suitable Models of Neurodegenerative Diseases (i) Huntington'sDisease

The genetically altered chondrocytes of the invention can be used toameliorate or modify neurodegeneration in a subject withneurodegenerative diseases such as Huntington's disease. Models ofHuntington's disease have been developed in several different animals,for example in the rat (Isacson et al. (1985) Neuroscience 16:799-817),monkey (Kanazawa, et al. (1986) Neurosci. Lett. 71:241-246), and baboon(Hantraye. et al. (1992) Proc. Natl. Acad. Sci. USA 89:4187-4191;Hantraye, et al. (1990) Exp. Neurol. 108:91-014; Isacson, et al.(1989)Exp. Brain Res. 75(1):213-220). These models of Huntington's diseasehave been described as providing effective therapeutic models that arepredictive of therapeutic efficacy in humans.

Neurodegeneration in Huntington's disease typically involvesdegeneration in one or both nuclei forming the striatum or corpusstratium, the caudate nucleus and putamen. Administration of thegenetically altered chondrocytes that express a therapeutic agent tothese regions may modify the function of these regions. The geneticallyaltered chondrocytes may be delivered to these regions using a liquidpolymer gel comprising the genetically altered chondrocytes, anddelivered using standard stereotactic delivery methods to the specificregions of the brain. The therapeutic effects of expressing thetherapeutic agent using genetically altered chondrocytes, can bedetermined by morphological and immunohistochemical studies. Behavioraltests can also be performed using standard techniques, such as the mazetest.

(ii) Parkinson's Disease

Parkinson's disease in humans primarily affects subcortical structures,especially the substantia nigra and loercus caeruleus. It ischaracterized by the loss of dopamine neurons in the substantia nigra,which have the basal ganglia as their major target organ. Several animalmodels of Parkinson's disease have been generated in which effectivetherapies are indicative of therapeutic efficacy in humans. These animalmodels include three rat models (the rats having lesions in substantianigral dopaminergic cells caused by treatment with 6-hydroxydopamine,1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), or surgicaltransection of the nigral striatal pathway) (See, e.g. Bjorklund et al.(1982) Nature 298:652-654), a rhesus monkey model (the monkeys havinglesions in substantia nigral dopaminergic cells caused by treatment withMPTP) (See, e.g., Smith, et al. (1993) Neuroscience 52):7-16; Bakay etal. (1985) Appl. Neurophysiol. 48:358-361; Zamir. et al. (1984) BrainRes. 322:356-360), and a sheep model (the sheep having lesions insubstantia nigral dopaminergic cells caused by treatment with MPTP)(Baskin, et al. (1994) Life Sci. 54:471-479). In one embodiment, thegenetically altered chondrocytes can be modified to express atherapeutic agent (e.g., GABA) that ameliorates, of modifies thefunction of cells associated with Parkinson's disease. The therapeuticeffect can be determined as described above for Huntington's disease.

b) Autoimmune Disorders (i) Diabetes

The genetically altered chondrocytes of the invention can be used toameliorate or modify autoimmune diseases such as diabetes. A summary ofinsulin-dependent diabetes mellitus and its animal models is describedby Wong et al. (1999) Curr Opin Immunol 11:643-647. A few autoantigenshave been associated with Type I diabetes mellitus, for example, insulin(Palmer et al. (1983) Science 222:1337-1339), glutamic aciddecarboxylase (GAD) (Baekkeskov et al. (1990) Nature 347:151-156) andcarboxypeptidase H (Castano. et al. (1991) J. Clin. Endocr. Metab,73:1197-1201), and the glycolipids GT3 (Gillard, et al. (1989) JournalImmunol. Methods 142:3826-3832) and GM2-1 (Dotta, et al. (1992)Endocrinology 130:37-42) and PM-1 (U.S. Pat. No. 5,908,627). Thesemodels can be used to investigate the effect of genetically alteredchondrocytes that express a therapeutic agent (e.g., insulin) ondiabetes in the animal.

(ii) Rheumatoid Arthritis

The genetically modified chondrocytes of the invention can also beimplanted in a patient to treat a disease such as Rheumatoid arthritis.In this embodiment, chondrocytes are implanted either in or close to thejoint capsule for the treatment of specific joint that is affected byRheumatoid arthritis, or in an ectopic site for a systemic delivery toall joints of an anti-inflammatory therapeutic agent. Examples of usefulanti-inflammatory therapeutic agents that can be expressed by thegenetically modified chondrocytes to treat Rheumatoid arthritis include,but are not limited to TNF-alpha antagonist and Remicade.

(c) Reproduction

Substances involved in reproduction can also be modified using thegenetically altered chondrocytes of the invention. Suitable animalmodels for reproduction are Sprague-Dawley rats, which are readilyavailable. For example, modifying the function of luteinizinghormone-releasing hormone (LHRH), a hormone regulated by thehypothalamus and involved in the stimulation of ovulation and uterinegrowth (Fueshko et al. (1994) Dev Biol 166:331-348; Hahn et al. (1984)Endocr Res, 10:123-138). Luteinizing hormone-releasing hormone alsoplays a role in male sterility by inhibiting the action of luteinizinghormone-releasing hormone with a synthetic decapeptide (Carelli (1982)Proc Natl. Acad. Sci. USA 79:5392-5395).

Alternatively, the genetically altered chondrocytes can be used todeliver estrogen for post-menopausal hormone therapy in women. Thesegenetically altered chondrocytes can be delivered to the uterus,fallopian tubes, ovaries and the like reproductive organs. Further,since diffusible factors such as hormones, need not be delivered to aspecific target site, unless there is a systemic toxic or unwanted sideeffect, alternative delivery options include local delivery accomplishedby injection through a syringe, surgical implantation, or endoscopicdelivery through a trochar. The genetically altered chondrocytes mayalso be used for sterilization purposes by delivering a sterilizationagent that causes sterility in males. The genetically alteredchondrocytes that express the sterilization agent can be delivered, forexample to the testis. Other male reproductive organs that can betargeted by the genetically altered chondrocytes include, but are notlimited to, testis, urethra, and ureter.

(d) Blood Related disorders

The genetically altered chondrocytes can be used to express therapeuticagents such as erythropoietin (EPO). EPO is a glycoprotein hormoneproduced primarily by cells of the peritubular capillary endothelium ofthe kidney, and is responsible for the regulation of red blood cellproduction. Secondary amounts of the hormone are synthesized in liverhepatocytes of healthy adults. In premature as well as full terminfants, the liver is the primary site of EPO production. The kidneybecomes the primary site of EPO synthesis shortly after birth. EPOproduction is stimulated by reduced oxygen content in the renal arterialcirculation. Circulating EPO binds to EPO receptors on the surface oferythroid progenitors resulting in replication and maturation tofunctional erythrocytes by an incompletely understood mechanism.

Clinical conditions that give rise to tissue hypoxia including anemia,lung disease, or cyanotic heart disease, lead to increased levels ofserum EPO. Low EPO levels are observed in patients with anemia, patientswith cancer, as well as those with rheumatoid arthritis, HIV infection,ulcerative colitis, and sickle cell anemia. Suppression of EPO synthesisby inflammatory cytokines (e.g., IL-1, TNF-alpha) is believed to occurin certain chronic diseases or cancer.

In contrast, elevation of EPO levels can occur in association with renaldiseases such as hydronephrosis or cysts, or certain tumors, resultingin erthrocytosis. Examples include renal cell carcinoma (hypernephroma),hepatocellular carcinoma, and adrenal gland tumors. Certain bone marrowdisorders, such as myelodysplastic syndrome and aplastic anemia, mayalso be associated with high serum levels of EPO. In the setting of bonemarrow disease, high serum EPO levels are presumably due to thereduction in the number of EPO receptor bearing cells, thereby allowingserum levels to rise. Thus, genetically modified chondrocytes can alsobe used to express EPO antibody to interact with the elevated EPO toreduce the level.

Suitable in an animal model of anemia that can be used to test thegenetically modified chondrocytes are available (See e.g., Hamamori etal., (1995), J. Clinical Investigation, 95:1808-1813, Osborne et al.,(1995) Proc. Natl. Acad. Sci., USA, 92:8055-8058). The geneticallymodified chondrocytes expressing EPO can be implanted into the anemicanimal and determining the level of EPO and the hematocrit of thetreated animal can be measured over time.

(e) Allergies

The genetically modified chondrocytes of the invention can also beimplanted in a patient to treat allergies by effecting the in vivoproduction of the allergens, or their antibodies through the geneticallymodified chondrocytes. Through controlled expression of the allergens,or their antibodies, the patient gradually develops IgG antibodies thatblock the IgE antibodies which result in long term relief fromallergies.

(f) Pain management

Pain management can be effected through the genetically modifiedchondrocytes as well by modifying the chondrocytes to express opiates orendophins. The genetically modified chondrocytes that express such painmediating substances would be implanted in tissue such as the brain. Inaddition, the chondrocytes can be modified to express nociceptive painrelievers, and the modified chondrocytes could be implanted locally, atthe site of the injury or disease causing pain.

(g) Cancer Treatment

Cancer is a disease that is characterized by uncontrolled growth ofabnormal or cancerous cells, in most instances as a result of an alteredgenome in a single abnormal cell. The alteration in the genome is causedby a mutation in one or more genes wherein the probability of theoccurrence of a mutation is increased by a variety of factors including(i) ionizing radiation, (ii) exposure to chemical substances known ascarcinogens, (iii) some viruses, (iv) physical irritation, and (v)hereditary predisposition.

The genetically modified chondrocytes can be used to deliver therapeuticagents that enhance a subject's immune response to invading metastasesor to either directly or indirectly suppress cancerous cell growth. Suchtherapeutic agents include, but are not limited to, various cytokinessuch as interleukin-2 (IL-2), granulocyte-macrophage colony stimulatingfactor (GM-CSF), interleukin-12 (IL-12) and interferon-gamma(IFN-gamma), anti-angiogenic molecules and tumor associated antigens(Anderson et al., (1990) Cancer Res., 50: 1853, Stoklosa, et al., (1998)Ann Oncol., 9:63, Leibson, et al., (1984) Nature, 309:799, Book, et al.,(1998) Semin. Oncol. 25:381, Salgaller, et al., (1998) J. Surg. Oncol.,68: 122, Griscelli, et al., (1998) Proc. Natl. Acad. Sci. USA, 95:6367).

Suitable models of tumors are available, for example, the mouse tumormodel (See e.g., Hearing et al., (1986) J. Iminunol., 137: 379).Therapeutic doses of anti-tumor molecules can be delivered in a mousetumor model using the genetically altered chondrocytes. In vivo tissueor serum levels of recombinant molecules are measured at varying timepoints following implantation and the effects on tumor development andanimal survival are followed over time. Other art-accepted animal modelsof cancer have been described in Hearing, (1986) J. Immunol., 137:379,Stoklosa et al., (1998) Ann. Oncol., 9:63, Carson et al., (1998) J.Surg. Res., 75:97, Maurer-Gebhard et al., (1998) Cancer Res., 58:2661and Takaori-Kondo et al., (1998) Blood 91:4747).

Therapeutic efficacy of treatment of cancer can be determined bymeasuring changes in clinical parameters such as tumor shrinkage (e.g.at least 5-10% and preferably 25-100%) and/or extended animal survivaltime.

(h) Solid Organ Treatment

The genetically modified chondrocytes can be used to deliver therapeuticagents (directly or in close proximity) to a region in a solid organsuch as the heart, kidney, or liver.

III. Biocompatible Substrates

The genetically altered chondrocytes of the invention can be deliveredto a desired environment by using biocompatible substrates. In oneembodiment of the present invention, the substrate can be formed from abiocompatible polymer. A variety of biocompatible polymers can be usedto make the biocompatible tissue implants or substrates according to thepresent invention. The biocompatible polymers can be synthetic polymers,natural polymers or combinations thereof. As used herein the term“synthetic polymer” refers to polymers that are not found in nature,even if the polymers are made from naturally occurring biomaterials. Theterm “natural polymer” refers to polymers that are naturally occurring.In embodiments where the substrate includes at least one syntheticpolymer, suitable biocompatible synthetic polymers can include polymersselected from the group consisting of aliphatic polyesters, poly(aminoacids), copoly(ether-esters), polyalkylenes oxalates, polyamides,tyrosine derived polycarbonates, poly(iminocarbonates), polyorthoesters,polyoxaesters, polyamidoesters, polyoxaesters containing amine groups,poly(anhydrides), polyphosphazenes, and blends thereof. Suitablesynthetic polymers for use in the present invention can also includebiosynthetic polymers based on sequences found in collagen, elastin,thrombin, fibronectin, starches, poly(amino acid), poly(propylenefumarate), gelatin, alginate, pectin, fibrin, oxidized cellulose,chitin, chitosan, tropoelastin, hyaluronic acid, ribonucleic acids,deoxyribonucleic acids, polypeptides, proteins, polysaccharides,polynucleotides and combinations thereof.

For the purpose of this invention aliphatic polyesters include, but arenot limited to, homopolymers and copolymers of lactide (which includeslactic acid, D-,L- and meso lactide); glycolide (including glycolicacid); ε-caprolactone; p-dioxanone (1,4-dioxan-2-one); trimethylenecarbonate (1,3-dioxan-2-one); alkyl derivatives of trimethylenecarbonate; δ-valerolactone; β-butyrolactone; γ-butyrolactone;ε-decalactone; hydroxybutyrate; hydroxyvalerate; 1,4-dioxepan-2-one(including its dimer 1,5,8,12-tetraoxacyclotetradecane-7,14-dione);1,5-dioxepan-2-one; 6,6-dimethyl-1,4-dioxan-2-one; 2,5-diketomorpholine;pivalolactone; α, α diethylpropiolactone; ethylene carbonate; ethyleneoxalate; 3-methyl-1,4-dioxane-2,5-dione;3,3-diethyl-1,4-dioxan-2,5-dione; 6,6-dimethyl-dioxepan-2-one;6,8-dioxabicycloctane-7-one and polymer blends thereof. Aliphaticpolyesters used in the present invention can be homopolymers orcopolymers (random, block, segmented, tapered blocks, graft, triblock,etc.) having a linear, branched or star structure.Poly(iminocarbonates), for the purpose of this invention, are understoodto include those polymers as described by Kemnitzer and Kohn, in theHandbook of Biodegradable Polymers, edited by Domb, et. al., HardwoodAcademic Press, pp. 251-272 (1997). Copoly(ether-esters), for thepurpose of this invention, are understood to include thosecopolyester-ethers as described in the Journal of Biomaterials Research,Vol. 22, pages 993-1009, 1988 by Cohn and Younes, and in PolymerPreprints (ACS Division of Polymer Chemistry), Vol. 30(1), page 498,1989 by Cohn (e.g., PEO/PLA). Polyalkylene oxalates, for the purpose ofthis invention, include those described in U.S. Pat. Nos. 4,208,511;4,141,087; 4,130,639; 4,140,678; 4,105,034; and 4,205,399.Polyphosphazenes, co-, ter- and higher order mixed monomer basedpolymers made from L-lactide, D,L-lactide, lactic acid, glycolide,glycolic acid, para-dioxanone, trimethylene carbonate and ε-caprolactonesuch as are described by Allcock in The Encyclopedia of Polymer Science,Vol. 13, pages 31-41, Wiley Intersciences, John Wiley & Sons, 1988 andby Vandorpe, et al in the Handbook of Biodegradable Polymers, edited byDomb, et al., Hardwood Academic Press, pp. 161-182 (1997).Polyanhydrides include those derived from diacids of the formHOOC—C₆H₄—O—(CH₂)_(m)—O—C₆H₄—COOH, where “m” is an integer in the rangeof from 2 to 8, and copolymers thereof with aliphatic alpha-omegadiacids of up to 12 carbons. Polyoxaesters, polyoxaamides andpolyoxaesters containing amines and/or amido groups are described in oneor more of the following U.S. Pat. Nos. 5,464,929; 5,595,751; 5,597,579;5,607,687; 5,618,552; 5,620,698; 5,645,850; 5,648,088; 5,698,213;5,700,583; and 5,859,150. Polyorthoesters such as those described byHeller in Handbook of Biodegradable Polymers, edited by Domb, et al.,Hardwood Academic Press, pp. 99-118 (1997).

As used herein, the term “glycolide” is understood to includepolyglycolic acid. Further, the term “lactide” is understood to includeL-lactide, D-lactide, blends thereof, and lactic acid polymers andcopolymers.

Elastomeric copolymers are also particularly useful in the presentinvention. Suitable elastomeric polymers include those with an inherentviscosity in the range of about 1.2 dL/g to 4 dL/g, more preferablyabout 1.2 dL/g to 2 dL/g and most preferably about 1.4 dL/g to 2 dL/g asdetermined at 25° C. in a 0.1 gram per deciliter (g/dL) solution ofpolymer in hexafluoroisopropanol (HFIP). Further, suitable elastomersexhibit a high percent elongation and a low modulus, while possessinggood tensile strength and good recovery characteristics. In thepreferred embodiments of this invention, the elastomer exhibits apercent elongation greater than about 200 percent and preferably greaterthan about 500 percent. In addition to these elongation and modulusproperties, suitable elastomers should also have a tensile strengthgreater than about 500 psi, preferably greater than about 1,000 psi, anda tear strength of greater than about 50 lbs/inch, preferably greaterthan about 80 lbs/inch.

Exemplary biocompatible elastomers that can be used in the presentinvention include, but are not limited to, elastomeric copolymers ofε-caprolactone and glycolide (including polyglycolic acid) with a moleratio of ε-caprolactone to glycolide of from about 35:65 to about 65:35,more preferably from 45:55 to 35:65; elastomeric copolymers ofε-caprolactone and lactide (including L-lactide, D-lactide, blendsthereof, and lactic acid polymers and copolymers) where the mole ratioof ε-caprolactone to lactide is from about 35:65 to about 65:35 and morepreferably from 45:55 to 30:70 or from about 95:5 to about 85:15;elastomeric copolymers of p-dioxanone (1,4-dioxan-2-one) and lactide(including L-lactide, D-lactide, blends thereof, and lactic acidpolymers and copolymers) where the mole ratio of p-dioxanone to lactideis from about 40:60 to about 60:40; elastomeric copolymers ofε-caprolactone and p-dioxanone where the mole ratio of ε-caprolactone top-dioxanone is from about from 30:70 to about 70:30; elastomericcopolymers of p-dioxanone and trimethylene carbonate where the moleratio of p-dioxanone to trimethylene carbonate is from about 30:70 toabout 70:30; elastomeric copolymers of trimethylene carbonate andglycolide (including polyglycolic acid) where the mole ratio oftrimethylene carbonate to glycolide is from about 30:70 to about 70:30;elastomeric copolymers of trimethylene carbonate and lactide (includingL-lactide, D-lactide, blends thereof, and lactic acid polymers andcopolymers) where the mole ratio of trimethylene carbonate to lactide isfrom about 30:70 to about 70:30; and blends thereof. Examples ofsuitable biocompatible elastomers are described in U.S. Pat. No.5,468,253.

In one embodiment, the elastomer is a copolymer of 35:65 ε-caprolactoneand glycolide, formed in a dioxane solvent and including a polydioxanonemesh. In another embodiment, the elastomer is a copolymer of 40:60ε-caprolactone and lactide with a polydioxanone mesh. In yet anotherembodiment, the elastomer is a 50:50 blend of a 35:65 copolymer ofε-caprolactone and glycolide and 40:60 copolymer of ε-caprolactone andlactide. The polydioxanone mesh may be in the form of a one layer thicktwo-dimensional mesh or a multi-layer thick three-dimensional mesh.

In another embodiment, the substrate can be in the form of an injectablegel matrix. The gel matrix can be a biological or synthetic hydrogel,including alginate, cross-linked alginate, hyaluronic acid, collagengel, fibrin glue, fibrin clot, poly(N-isopropylacrylamide), agarose,chitin, chitosan, cellulose, polysaccharides, poly(oxyalkylene), acopolymer of poly(ethylene oxide)-poly(propylene oxide), poly(vinylalcohol), polyacrylate, platelet rich plasma (PRP) clot, platelet poorplasma (PPP) clot, Matrigel, or blends thereof.

The gel matrix is selected to have properties that allow the therapeuticagent to be expressed within the gel matrix, as well as allowing theexpressed therapeutic agent to diffuse out of the gel matrix into thesurrounding environment.

Examples of gel matrices that may be used include, but are not limitedto, collagen gel, fibrin glue, polyglycolic acid, polylactic acid,polyethylene oxide gel, alginate or calcium alginate gel,poly-(2-hydroxyethyl methacrylate) (i.e., a hydrogel), polyorthoester,hyaluronic acid, polyanhydride, chitosan, gelatin, agarose, and otherbioresorbable and biocompatible materials such as those described in EP0705878 A2. To promote chondrocyte proliferation and function, thebiological gel can additionally contain appropriate nutrients (e.g.,serum, salts such as calcium chloride, ascorbic acid, and amino acids)and growth factors (e.g., somatomedin, basic fibroblast growth factor,transforming growth factor-β, cartilage growth factor, bone-derivedgrowth factor, or a combination thereof).

Polysaccharides are a class of macromolecules of the general formula(CH₂O)n which are also useful as the hydrogel substrate in the presentinvention. Polysaccharides include several naturally occurringcompounds, e.g., agarose, alginate and chitosan.

Agarose is a clear, thermoreversible hydrogel made of polysaccharides,mainly the alternating copolymers of 1,4 linked and3,6-anhydro-α-L-galactose and 1,3 linked β-D-galactose. Two commerciallyavailable agaroses are SeaPrep™ and SeaPlaque™ agarose (FMC Corp.Rockland, Me.). The thermoreversible properties of agarose gels make itpossible for agarose to be a liquid at room temperature allowing foreasy mixing of cell-gel solution and then cooling to 4° C. causesgelation and entrapment of cells. This is a comparatively benignprocess, free of chemicals toxic to the cells.

The agarose concentration can be about 0.50 to 2% (w/v), most preferablyabout 1.0%. In any event, the concentration of agarose should besufficient to permit chondrocyte encapsulation at a concentration thatvaries in the range of about 10⁴ cells/ml to 10⁷ cells/ml, and morepreferably in the range of about 10⁵ to 10⁶ cells/ml.

Alginate is a carbohydrate polymer isolated from seaweed, which can becrosslinked to form a hydrogel by exposure to a divalent cation such ascalcium, as described, for example in WO 94/25080, the disclosure ofwhich is incorporated herein by reference. The modified alginatesolution is mixed with the cells to be implanted to form a suspension.Then the suspension is injected directly into a patient prior tocrosslinking of the polymer to form the hydrogel containing the cells.The suspension then forms a hydrogel over a short period of time due tothe presence in vivo of physiological concentrations of calcium ions(See e.g., U.S. Pat. No. 4,352,883 to Lim, incorporated herein byreference).

In general, the polymers that make up the biological gels are at leastpartially soluble in aqueous solutions, such as water, buffered saltsolutions, or aqueous alcohol solutions. Methods for the synthesis ofthe polymers are known to those skilled in the art (See e.g., ConciseEncyclopedia of Polymer Science and Polymeric Amines and Ammonium Salts,E. Goethals, editor (Pergamen Press, Elmsford, N.Y. 1980)). Naturallyoccurring and synthetic polymers may be modified using chemicalreactions available in the art and described, for example, in March,“Advanced Organic Chemistry,” 4th Edition, 1992, Wiley-IntersciencePublication, New York.

Water soluble polymers with charged side groups may be crosslinked byreacting the polymer with an aqueous solution containing ions of theopposite charge, either cations if the polymer has acidic side groups oranions if the polymer has basic side groups. Examples of cations forcrosslinking of the polymers with acidic side groups to form a hydrogelare monovalent cations such as sodium, and multivalent cations such ascopper, calcium, aluminum, magnesium, strontium, barium, and tin, anddi-, tri- or tetra-functional organic cations such as alkylammoniumsalts. Aqueous solutions of the salts of these cations are added to thepolymers to form soft, highly swollen hydrogels and membranes. Thehigher the concentration of cation, or the higher the valence, thegreater the degree of cross-linking of the polymer. Additionally, thepolymers may be crosslinked enzymatically, e.g., fibrin with thrombin.

Several physical properties of the biological gels are dependent upongel concentration. Increase in gel concentration may change the gel poreradius, morphology, or its permeability to different molecular weightproteins. Gel pore radius determination can be effected by any suitablemethod, including hydraulic permeability determination using a graduatedwater column, transmission electron microscopy and sieving spheres ofknown radius through different agar gel concentrations (See, e.g.,Griess et al., (1993) Biophysical J., 65:138-48).

A variety of other suitable biological gels are also known. The polymercan be mixed with cells for implantation into the body and can becrosslinked to form a hydrogel matrix containing the cells either beforeor after implantation in the body. A hydrogel is defined as a substanceformed when an organic polymer (natural or synthetic) is crosslinked viacovalent, ionic, or hydrogen bonds to create a three-dimensionalopen-lattice structure which entraps water molecules to form a gel.

One skilled in the art will appreciate that the volume or dimensions(length, width, and thickness) of the biological gel comprising thechondrocytes can be selected based on the region or environment intowhich the biological gel substrate is to be implanted. In oneembodiment, the biological gel has a length (defined by a first andsecond long end) of about 10 cm to about 30 cm. The biological gel canfurther have a length of about 15 cm to about 25 cm. In a preferredembodiment, the biological gel has a length of about 20 cm. In anotherembodiment, the biological gel has a width (defined by a first andsecond short end) of about 0.5 cm to about 4.0 cm. The biological gelcan further have a width of about 1.0 cm to about 3.0 cm. In a preferredembodiment, the biological gel has a biocompatible substrate with widthof about 2.0 cm.

IV. Manufacturing of Therapeutic Agents Using Genetically AlteredChondrocytes

In another aspect, the invention pertains to using the geneticallyaltered chondrocytes for large scale in vitro preparation of therapeuticagents. For example, liter scale production (e.g., several liters) couldbe effected with expansion and maintenance of chondrocytes onmicrocarrier beads. Chondrocyte cells could be genetically manipulatedto express a therapeutic molecule of commercial value, then expanded andseeded onto microcarrier-bead containing bioreactor. Alternatively,chondrocytes could be seeded directly onto microcarrier beads andexpanded directly in bioreactors.

The following examples are illustrative of the principles and practiceof this invention. Numerous additional embodiments within the scope andspirit of the invention will become apparent to those skilled in theart.

EXAMPLES Example 1 In Vitro Isolation and Culturing of Chondrocyte Cells

This example describes one of many possible methods of isolating andculturing chondrocyte cells. Articular cartilage was asepticallyobtained from calf knee joints. Cartilage was shaved from the end of thebones using a scalpel. The tissue was finely minced in saline solutionand submitted to collagenase and trypsin digestion for several hours at37° C. until the tissue was completely digested and single cells weregenerated. The chondrocyte cells isolated from the cartilage tissue werethen washed from enzyme and seeded onto tissue culture plastic dishes ata concentration of about 10,000 cells/cm². The chondrocyte cells whereexpanded in 10% FBS containing DMEM culture medium. Following expansion,the chondrocytes can be released from the vessel using a trypsin/EDTAtreatment, centrifuged and concentrated or diluted to a desiredconcentration to achieve for example a seeding density of approximately5,000 cells to about 10,000 cells per square centimeter when plated ontoculture dishes. Alternatively, the expanded chondrocytes cells can befrozen according to standard procedures until needed.

Example 2 In Vitro Transfection of Chondrocyte Cells and Expression of aMarker Protein

This example described the techniques used to transfect chondrocyteswith a marker protein, Green fluorescent protein (GFP). A DNA vectorcarrying a reporter gene is used to establish proof of concept for theutility of normal articular chondrocytes as a drug delivery system fortherapeutic application in ectopic sites. As a model, a DNA vector(pTracer-SV40) carrying a gene for green fluorescent protein was used asa surrogate to model a protein not normally present in chondrocytes, andof therapeutic value. If chondrocytes can be transfected by the modelvector and expressed the foreign protein, it would demonstrated theability of using this system for therapeutic protein. In the followingexample, a lipid formulation from Stratagene (GeneJammer) was used tointroduced the foreign DNA into chondrocytes. Any other reagent orphysical means of introducing DNA into cell would serve the samepurpose.

Exponentially growing chondrocytes were seeded onto 35 mm dishes at adensity of 1.5 to 3.7×10⁶ cells in 10% FBS containing DMEM. In separateplastic tube, serum-free medium was combined with transfection reagent(GeneJammer from Stratagene) and incubated with the chondrocytes for5-10 minutes. Plasmid DNA (pTracer-SV40 from Invitrogen) was mixed withthe chondrocytes, and the mixture further incubated for 5-10 minutes.The transfection mixtures was then added to the 35 mm dishes andincubated for at least 3 hours at 37° C. Fresh media (10% FBS containingDMEM) was then added and the cells are fed every 2-3 days with freshserum containing media.

In order to identify transfected chondrocytes, the culture chondrocytecells were observed under fluorescent light. To facilitate this, theculture media was removed and replaced with phosphate buffered salinesolution. The cultured chondrocyte cells were examined by fluorescencemicroscopy. Positive chondrocyte cells were identified as those thatincorporated the plasmid, expressed GFP, and fluoresced green under themicroscope, as shown in FIG. 2.

The results illustrate the ability of introducing and expressing aforeign protein in articular chondrocytes. These results further showthat chondrocytes provide a robust, stable cell for expression of aheterologous protein.

Example 3 In Vitro Transfection of Human and Bovine Chondrocyte Cellsand Expression of EPO Protein

This example describes the techniques used to transfect chondrocyteswith a therapeutic agent, human erythropoietin (EPO).

An EPO vector was generated which comprised the EPO gene (GenbankAccession No. 182198) inserted into the commercially available pSG5backbone (Stratagene Inc.). The pSG5 plasmid contains ampicillin andkanamycin as a selection marker for bacteria. This construct alsocontains F1 filamentous phage origin and SV40 promoter for betterexpression. The 584 base pair EPO fragment was cloned into the NCO/BamH1 site site of pSG5. The size of this vector was about 4.6 kb.

A second EPO vector was generated which comprised the EPO gene insertedinto the commercially available pcDNA3.1 backbone (Invitrogen LifeTechnologies). This particular construct has the antibiotic Neomycin asa selection marker for mammalian cells.

Each of the EPO vectors was transfected into human chondrocytes usingFuGENE 6, a commercially available lipid transfection agent (RocheDiagnostics Corporation). Primary human chondrocytes were passaged once,then seeded onto 35 mm plates at a density of 250,000 cells/plate in 10%FBS containing DMEM. The transfection reagents were combined in serumfree medium at a ratio of 3 μl FuGENE to 1 μg plasmid and incubated for15 minutes at room temperature. The resulting transfection mixture wasthen added dropwise to the chondrocyte plates, with a total of 1.0 μg ofEPO plasmid added to each plate. The cell cultures were incubated at 37°C. Supernatant was collected, in aliquots of 0.5 ml, at 20, 48, 72 and96 hours and stored at 4° C.

For this experiment, two control samples were prepared. The firstcontrol comprised chondrocytes which were transfected with a geneencoding an EPO mimetibody. The second control comprised cells to whichno reagents were added. The above transfection procedure was repeatedfor human and bovine chondrocytes, with similar cell density, reagentconcentrations and control conditions.

Expression of EPO was determined by measuring the supernatant EPOconcentration synthesized by the chondrocytes using a commerciallyavailable ELISA kit which is specific to EPO (Quantikine IVD, R&DSystems Inc.). Absorption at 450 nm was measured to determine the amountof bound protein on the ELISA plates.

FIGS. 3 a and b shows that, for both human and bovine chondrocytes,neither control samples exhibited significant EPO expression, while bothof the EPO transfection constructs resulted in significant EPOexpression. Expression of human EPO by both human and bovinechondrocytes demonstrates the feasibility of allogeneic and xenogenicapproaches to chondrocyte-based in vivo therapeutic delivery.

Example 4 In Vitro Transfection of Human Chondrocyte Cells andExpression of EPO Mimetibody

This example described the techniques used to transfect chondrocyteswith a therapeutic agent, the EPO mimetibody (mEPO). This compound isdesigned to mimic the binding region of the EPO protein, therebyproviding the same therapeutic function as EPO when it attaches to theEPO receptor and activates it.

A construct was developed comprising the mEPO gene inserted into thecommercially available pcDNA3.1 backbone (Invitrogen Life Technologies).This particular construct has the antibiotic, Neomycin as a selectionmarker for mammalian cells, and Ampicillin as a selection marker forbacteria. The size of this vector construct was about 6 kb. Anotherconstruct was developed comprising the mEPO gene inserted into thecommercially available pCEP4 backbone (Invitrogen Life Technologies).This construct has hygromycin as a selection marker for mammalian cells,and contains EBV replication origin and nuclear antigen (EBNA-1) forbetter expression. The size of this vector is about 11 kb.

Each plasmid encoding mEPO was transfected into human chondrocytes usingthe method outlined in Example 3. Control samples comprised chondrocytestransfected with human EPO, as well as cells to which no reagents wereadded.

Expression of mEPO was measured using an ELISA which is specific to thehuman IgG region of the mimetibody. After 96 hours of transfection, EPOmimetibody expression was detected at a level of 18 ng/ml. As shown inFIG. 4, this expression level was substantially higher than the baselinemEPO level measured for control samples. Collectively, these resultsdemonstrate that chondrocytes can be used to express a therapeuticagents such as EPO and mEPO.

Example 5 Creating Chondrocyte-Gel Substrates

This Example describes how to formulate biological gel matrix substratescomprising genetically altered chondrocytes that express a therapeuticagent. Chondrocytes growing in monolayer (FIG. 5A) were released fromthe culture dish using trypsin/EDTA. The cells were counted andsuspended in low melting agarose at 40° C. prepared in serum free mediumsuch that the final concentration of cells was 100,000/ml of agarosegel. 3 ml of gel was dispensed into a 30 mm dish and allowed to set atroom temperature. The gel was then overlayed with an equal volume of 10%FBS containing culture medium. The media is replenished every 2 to 3days and cultures maintained for several weeks. After a few weeks,colonies of differentiating chondrocytes can be observed (FIG. 5B). Thegels were fixed in formalin 6 weeks after initial suspension and stainedwith alcian blue. Cartilage matrix deposited by the chondrocytes stainsblue and indicates cartilage differentiation (FIG. 5C). Monitoring oftherapeutic protein expression could be performed by sampling theculture media overtime. Establishing a delivery dose from such agarosesuspended chondrocytes could be done by harvesting different size disksgenerated for example with a biopsy punch.

Example 6 Use of Chondrocytes to Express a EPO or mEPO in a Non-TypicalChondrocyte Environment for a Blood Disorder

This example describes the use chondrocytes that have been geneticallyaltered to express EPO or mEPO in an in vivo system. The chondrocyteswere genetically altered as described in Examples 3 and 4 to express EPOor mEPO, and placed into biological gel matrix substrates as describedin Example 5. Slices of the solid gel, or a liquid gel that solidifieson contact, can be placed into an animal model of anemia (See Osborn etal, Supra), and the amelioration of anemia was observed by measuring thehemocrit levels of the blood.

Example 7 Use of Chondrocytes to Express Therapeutic Agent in aNon-Typical Chondrocyte Environment for an Autoimmune Disorder

This example describes the use chondrocytes that have been geneticallyaltered to express a therapeutic agent for amelioration of an autoimmunedisorders, such as expressing insulin an animal model for diabetes.

The chondrocytes were genetically altered as described in Example 2 toexpress the insulin gene, and placed into biological gel matrixsubstrates as described in Example 5. Slices of the solid gel, or aliquid gel that solidifies on contact, can be placed (e.g., into thepancreas) in an animal model of insulin (See e.g., Wong et al. Supra),and changes in the blood glucose levels can be observed.

Example 8 Use of Chondrocytes to Express a Gaba in a Non-TypicalChondrocyte Environment for Neurodegenerative Disorder

This example describes the use chondrocytes that have been geneticallyaltered to express GABA an animal model for Parkinson's disease. Thechondrocytes were genetically altered as described in Example 2 toexpress GABA, and placed into biological gel matrix substrates asdescribed in Example 5. Slices of the solid gel, or a liquid gel thatsolidifies on contact, can be placed into a region of the brain (e.g.,subthalmic nucleus) in a model for Parkinson's disease (See e.g.,Bjorklund et al., Supra), and the amelioration of Parkinson's symptomscan be observed be monitoring the behavioral changes in the animal(e.g., by a maze test).

One skilled in the art will appreciate further features and advantagesof the invention based on the above-described embodiments. Accordingly,the invention is not to be limited by what has been particularly shownand described, except as indicated by the appended claims. Allpublications and references cited herein are expressly incorporatedherein by reference in their entirety.

1. A method for ameliorating a disorder or injury in a subject using agenetically altered chondrocyte, comprising: providing a geneticallyaltered chondrocyte, wherein the genetically altered chondrocyte hasbeen altered to express a therapeutic agent; implanting a biocompatiblesubstrate comprising a genetically altered chondrocyte into a targetregion of the subject, wherein the genetically altered chondrocyte isnot structurally functional in the target region or an environmentsurrounding the target region; and expressing the therapeutic agent inthe target region at a level sufficient to ameliorate the disorder. 2.The method of claim 1, wherein the target region is in an atypicalchondrocyte environment.
 3. The methods of claim 2, wherein the atypicalchondrocyte environment is in an organ selected from the groupconsisting of the brain, heart, liver, kidney, gastro-intestinal tract,spleen, smooth and skeletal muscles, eye, ganglions, lungs, gonads, andpancreas.
 4. The methods of claim 2, wherein the atypical chondrocyteenvironment is an aqueous environment selected from the group consistingof blood and plasma.
 5. The method of claim 1, wherein the target regionis in a typical chondrocyte environment.
 6. The method of claim 5,wherein the typical chondrocyte environment is selected from the groupconsisting of bone, tendon, and cartilage.
 7. The method of claim 1,wherein the step of implanting the biocompatible substrate comprisesimplanting a gel matrix substrate.
 8. The method of claim 7, wherein thegel matrix substrate is selected from the group consisting of alginate,polysaccharide, and agarose.
 9. The method of claim 7, wherein thedimensions of the implanted gel matrix substrate determines theconcentration of chondrocytes within the gel matrix substrate that areavailable to express the therapeutic agent.
 10. The method of claim 9,wherein the concentration of chondrocytes in the gel matrix substrate isabout 100,00 to 10 millions cells per mil in a gel matrix volume of 0.05ml to 10 ml.
 11. The method of claim 1, wherein the disorder is selectedfrom the group consisting of a blood disorder, an autoimmune disorder, ahormonal disorder, an anti-inflammatory disorder, a fertility disorder,and an neurodegenerative disorder.
 12. The method of claim 1, whereinthe injury is selected from the group consisting of a wound, a bonedefect, a cartilage defect, a skin wound, and a torn ligament.